HIV enters human T cells through the fusion of viral and host-cell membranes; this process is mediated by a viral surface protein, gp41, and by the platform provided by the cholesterol-rich viral membrane. The fusion process is a major target in vaccine design as well as for developing new therapeutics. To date, extensive efforts have focused on determining the structure and function of the virion surface proteins, such as gp41. However, there are significant gaps in understanding the function of the HIV membrane and its interactions with gp41 during viral entry and membrane fusion. The central hypothesis of this work is that the membrane-bound region of gp41 facilitates HIV infection through its interaction with the cholesterol-rich viral membrane, and that this interaction is disrupted by anti-gp41 neutralizing antibodies. This hypothesis will be investigated via the following specific aims: 1). To understand the membrane interactions of the membrane proximal ectodomain region (MPER) of gp41, including how the MPER exerts its fusogenic activity by perturbing membrane integrity, how this activity is affected by cholesterol, and how the MPER interacts with cholesterol in the viral membrane through its functional groups, such as its cholesterol recognition/interaction amino acid consensus (CRAC) motif; 2). To elucidate how anti-gp41 neutralizing antibodies disrupt MPER-membrane interactions, including how lipid composition affects antibodies binding to the MPER of gp41, and how those antibodies abolish the membrane-perturbing activity of the MPER and inhibit its interactions with cholesterol. The approach in this work is innovative because it will develop new electron paramagnetic resonance (EPR) techniques to explore the mechanism of HIV infection from a novel perspective - the role of the cholesterol-rich viral membrane and protein-membrane interaction. In particular, this work will be greatly facilitated by the unique, high-sensitivity EPR spectrometers at the National High Magnetic Field Laboratory and Florida State University. The proposed research is expected to have a significant impact on the current understanding of the roles of the viral membrane and surface proteins during viral infection and antibody neutralization. This knowledge will be directly applicable to the development of gp41-based immunogens and viral-fusion inhibitors.